LC/QTOF Identification of

Microcystin Variants in Cell-

Bound Algae from the San

Francisco Estuary

Ralph Hindle

Vogon Laboratory Services Ltd.

Cochrane, Alberta, Canada

Max Mizel

Dept. of Civil and Environmental Engineering

UC Davis

Davis, California

1

• Investigate impact of Microcystis aeruginosa in

San Francisco Estuary Delta in summer/fall of

2014

• Magnitude

• Timing

• Distribution

• Toxin content (cell-bound)

• Identify and quantitate MC-LR

• Identify other MC’s that may be present

• Nitrogen source

• Food web impact

Project Outline

Sampling Sites

3

General Microcystin Structure

> 70 microcystins have been identified in the literature

Microcystin

Examples

X Z Formula Neutral Mass

LR Leucine Arginine C49H74N10O12 994.5488

Desmethyl LR Leucine Arginine C48H72N10O12 980.5331

RR Arginine Arginine C49H75N13O12 1037.5658

YR Tyrosine Arginine C52H72N10O13 1044.5280

LW Leucine Phenylalanine C54H72N8O12 1024.5270

LF Leucine Tryptophan C52H71N7O12 985.5161

HtyR Homotyrosine Arginine C53H74N10O13 1058.5437

QQQ MRM Parametersa

5

a These patterns are useful, as they show that all supplied MCs fragment to the same 2 diagnostic ions

b RR readily shows a doubly-charged precursors. Depending on source conditions, other MCs can show either singly- or doubly-charged ions.

Microcystin Precursor b

(m/z)

Product Ion

(m/z)

Collision Energy

(V)

LR 995.6 135.2

213.2

80

80

Desmethyl-LR 981.5 135.2

213.2

80

80

RR 520.0 135.2

213.2

30

40

YR 1045.5 135.2

213.2

80

70

LW 1025.5 135.2

213.2

80

60

LF 986.5 135.2

213.2

70

50

HtyR 1059.5 135.2

213.2

80

70

Common Microcystin Fragments

m/z = 135.1 and 213.1

C9H11O+

m/z = 135.0804

Glu + Mdha = C9H13N2O4+

m/z = 213.0870

Zoom in on common MC fragments - 135

135.0804 = C9H11O+

• De-methylation is common in microcystins• Two 135 ions are present from 2 different

fragments• Indicates that Adda group is intact and not

de-methylated

135.1168 = C10H15+

C9H11O+

C10H15+

LC-QTOF Instrumentation

8

HPLC Mass Spectrometer

1290 Binary Pump 6520 or 6540 QTOF

1200 Degasser Electrospray Source

1290 Autosampler

1290 TCC

Database Score

Monoisotopic mass(varies in ppm)

Isotope spacing(varies in ppm)

Isotope distribution(varies in %)

Score based on:

• Mass match – measured versus given.

• Abundance score – abundance pattern of measured isotope cluster compared withvalues predicted from the proposed formula.

• Spacing match – how the m/z spacingbetween the lowest m/z ionand the A+1 and A+2 ions compared withthe values predicted from the proposed formula.

Chromatography

Mass tolerance 5 ppm; EIC extraction 50 ppm; spectral average < 10% saturation

12

Product Ion Spectrum for MC-LR

Score = 97.7Acc = 1.9 ppm

13

Product Ion Spectrum for MC-RR

Score = 96.6Acc = 2.3 ppm

14

Product Ion Spectrum for MC-LA

Score = 95.0Acc = 1.0 ppm

MC-LR Desmethylation Sites

Arg4

Leu2

MeAsp3

Ala1

Mdha7Glu6

Adda5

C49H74N10O12

Mass = 994.5488C48H72N10O12

Mass = 980.5331

- CH2

- 14.0157

Comparison of IC50 for MC Variants

17

MC Variant IC50 (µg/mL)

[Asp3, Z-Dhb7] MC-LR 0.05

[Asp3, Z-Dhb7] MC-HtyR 0.12

[Asp3, E-Dhb7] MC-LR 0.13

[Asp3, Dha7] MC-LR 0.22

[Asp3] MC-LR 0.22

[Dha7] MC-LR 0.22

[Asp3, E-Dhb7] MC-HtyR 0.33

[Asp3] MC-HtyR 0.35

[Dha7] MC-YR 0.42

MC-LR 0.80

MC-YR 1.48

[Asp3, Dha7] MC-RR 4.11

[Asp3, E-Dhb7] MC-RR 4.95

[Dha7] MC-RR 5.33

[Asp3] MC-YR > 10

MC-RR > 10

IC50 = Inhibitory Concentration for cytotoxicity, from Shimizu et al, Toxins 2014, 6, 168-179

Trp200 His191

Gln122

Val126Tyr127Tyr265

Cys269

Arg89

Arg214

Ile123

Glu

53

X-ray crystal structures of MC-LR bound to the catalytic domain of PP2A. The data is from the protein database on the net, provided as part of Xing, Y., Xu, Y., Chen, Y., Jeffrey, P. D., Chao, Y., Lin, Z., Li, Z., Strack, S., Stock, J. B., and Shi, Y. (2006) Structure of protein phosphatase 2A core enzyme bound to tumor-inducing toxins. Cell 127, 341–353. The picture was generated by Christopher Miles (Oslo, Norway) using Pymol.

Fragment Without Desmethylation

20

ASP3 or dm-Adda5

21

Desmethylation of Adda5

22

dm-Adda5

23

40 Microcystins Found in Trial Sample

24

Name Formula Mass Score

MC-LA C46 H67 N7 O12 909.4856 95.0

MCYST-AR C46 H68 N10 O12 952.5013 92.6

MCYST-AR C46 H68 N10 O12 952.5025 99.3

[D-Asp3,Glu(OMe)]MCYST-LAba C47 H69 N7 O12 923.4992 92.5

MCYST-LAba C47 H69 N7 O12 923.5007 99.7

[D-Asp3, Dha7]MCYST-LR C47 H70 N10 O12 966.5176 98.9

MCYST-ER C48 H70 N10 O14 1,010.5105 90.1

[D-Asp3]MCYST-LL C48 H71 N7 O12 937.5156 99.4

[D-Asp3]MCYST-LL C48 H71 N7 O12 937.5155 99.1

MCYST-LM C48 H71 N7 O12 S 969.4874 97.8

[DMAdda5]MCYST-LR C48 H72 N10 O12 980.5307 92.2

[DMAdda5]MCYST-LR C48 H72 N10 O12 980.5325 92.0

MCYST-MR C48 H72 N10 O12 S 1,012.5051 98.7

MCYST-M(O)R C48 H72 N10 O13 S 1,028.5012 95.2

[D-Asp3]MCYST-RR C48 H73 N13 O12 1,023.5512 96.5

MCYST-FA C49 H65 N7 O12 943.4704 98.3

MCYST-YAba C49 H65 N7 O13 959.4631 90.4

MCYST-LL C49 H73 N7 O12 951.5311 97.8

LR C49 H74 N10 O12 994.5505 98.9

RR C49 H75 N13 O12 1,037.5682 96.6

Name Formula Mass Score

[L-Mser7]MCYST-LR C49 H76 N10 O13 1,012.5583 90.2

MCYST-HilR C50 H76 N10 O12 1,008.5644 99.0

MCYST-WA C51 H66 N8 O12 982.4811 99.0

MCYST-NfkA C51 H66 N8 O14 1,014.4684 97.1

[Asp3]MCYST-LF C51 H69 N7 O12 971.4998 97.0

[D-Asp3]MC-FR C51 H70 N10 O12 1,014.5179 95.5

[D-Asp3]MCYST-(H4)YR C51 H74 N10 O13 1,034.5428 97.9

[D-Asp3]MCYST-(H4)YR C51 H74 N10 O13 1,034.5440 97.8

MCYST-WAba C52 H68 N8 O12 996.4963 99.6

LF C52 H71 N7 O12 985.5163 92.2

MCYST-FR C52 H72 N10 O12 1,028.5335 98.2

[D-Asp3]MCYST-HtyR C52 H72 N10 O13 1,044.5269 98.2

MCYST-(H4)YR C52 H76 N10 O13 1,048.5595 98.9

[D-Asp3]MCYST-LW C53 H70 N8 O12 1,010.5105 97.9

[DMAdda5]MC-Y(OMe)R C53 H74 N10 O14 1,074.5385 93.6

LW C54 H72 N8 O12 1,024.5264 98.7

LW C54 H72 N8 O12 1,024.5273 92.9

MCYST-WR C54 H73 N11 O12 1,067.5446 98.8

LR-GSH C59 H91 N13 O18 S 1,301.6327 91.8

RR-GSH C59 H92 N16 O18 S 1,344.6510 96.2

MC Variants – All Sites

25

26

Putative MC-WR MS/MS Spectrum

C9H11O+

C10H15+

Glu + Mdha = C9H13N2O4+

m/z = 213.0870

Microcystins Reviewed

27

dm-RR FA FR LR MC-LA RR WA WR

5 15 12 25 36 175 18 27

2 5 15 23 9 63 7 28

3 1 3 120 5 93 1 6

4 0 2 26 1 167 1 3

4 32 63 122 85 149 28 81

4 0 1 25 1 123 0 2

4 21 30 71 45 176 22 47

5 28 50 105 52 238 30 71

5 1 3 19 1 135 1 7

2 0 3 17 2 80 1 8

10 0 2 23 - 268 0 9

4 - 1 7 - 95 1 4

3 0 2 29 - 115 1 5

4 - 1 34 - 184 - 3

4 - 1 18 - 129 - 4

6 - 2 47 0 281 0 7

17 - 11 49 3 439 1 33

6 - 4 14 1 154 1 13

0 0 0 6 - 0

0 0 0 0 - 2 0 0

0 0 0 - 1 - 0

• 40 microcystin variants were detected in a single San

Francisco Delta algae sample immediately preceding

study• Accurate mass search of database with 123 unique chemical formulas

• Mass identification scores > 90

• Mass accuracy within 2 ppm

• MS/MS spectra for putative compounds without standards are being

reviewed

• Semi-quantitation will be done using response factors for known

standards

Summary

Acknowledgements

• California Department of Water Resources

• Tomo Kurobe (Project Manager)• UC Davis, Davis, CA

• Swee Teh (data release)• UC Davis, Davis, CA

• Christopher Miles (MC database)• Norwegian Veterinary

Institute (Oslo)

• Joe Weitzel (Travel to UC Davis)• Global Marketing Manager

(Agilent - USA)